1. Field of the Invention
The present invention relates to analyzing samples using evanescent wave biosensors. More specifically, the present invention relates to detecting single nucleotide polymorphisms in DNA samples using evanescent wave biosensors.
2. State of the Art
Over the past decade, molecular biology has been used to understand the molecular bases of inherited diseases. By identifying the gene or genes responsible for a disease, the genes of an afflicted person are compared to those of a non-afflicted person. In many diseases, the only difference between the genes of the afflicted and non-afflicted person is a single nucleotide polymorphism (“SNP”) in the DNA sequence. Based on this SNP or mutation, it is sometimes possible to screen for the disease. Some diseases that have been investigated by molecular biologists include inherited cardiovascular diseases, such as arrhythmogenic right ventricular dysplasia, familial hypertrophic cardiomyopathy, idiopathic ventricular fibrillation, long-QT syndrome and Marfan Syndrome. Of these cardiovascular diseases, familial hypertrophic cardiomyopathy (“HCM”), long-QT syndrome (“LQTS”), and Marfan Syndrome (“MFS”) are the best understood at the molecular level. Inheritance of these diseases is autosomal dominant and affected individuals are at risk of sudden cardiac death, often without previous symptoms. Since many of the genes responsible for these cardiac disorders, and a number of mutations in those genes, have been identified, it may be possible to use molecular diagnosis to screen individuals who may be at risk for sudden cardiac death.
Four genes have been implicated in LQTS, including KVLQT1, HERG, SCN5A, and KCNE1. Numerous mutations in these genes have been cataloged, including 75 mutations in KVLQT1, 84 mutations in HERG, 8 mutations in SCN5A, and 7 mutations in KCNE1. Of these four genes, the KVLQT1 gene is of great interest because it has been implicated in nearly 50% of the observed cases of LQTS in which the affected individual has been genotyped, a significant number of missense and deletion mutations have been identified in this gene by sequencing DNA from affected individuals, and the development of high-throughput screening assays for this gene may have a significant impact on treatment and patient outcomes. KVLQT1 encodes for a potassium channel in cardiac muscle, or at least the alpha subunit of the channel. Numerous SNPs occurring in KVLQT1 have been associated with LQTS. The KVLQT1 gene comprises 16 exons that range in size from 47 base pairs for exon 14 to 386 base pairs for exon 1. A disproportionate number of these SNPs have been observed in exon 7, suggesting that exon 7 may be a mutational hot spot. However, SNPs have been observed in other exons. One polymorphism is G760A, which occurs in exon 3 of KVLQT1, in which guanine (G) at position 760 in the nucleotide sequence is replaced by adenine (A). This mutation results in the substitution of methionine for valine at position 254 in the amino acid sequence of the KVLQT1 protein.
Assessing polymorphisms in humans currently involves isolating the gene of interest from afflicted individuals using polymerase chain reaction (“PCR”), sequencing the genes, and then cataloging any observed polymorphisms or mutations. However, this procedure is too expensive and time-consuming to be used in routine patient screening. These disadvantages led to the development of DNA chips that contain hundreds or thousands of nucleic acid probe molecules immobilized to a single substrate in a two-dimensional array. These nucleic acid probes correspond to known mutations, such as missense mutations or deletions, which have already been cataloged. The nucleic acid probes are known as allele-specific oligonucleotides (“ASO”). However, patient screening with the DNA chip still involves isolating and amplifying the gene(s) of interest from the patient's DNA using PCR and then allowing the PCR product(s) to bind to the DNA chip. The chip is then washed and DNA hybridization is detected, usually by fluorescence, using either an epifluorescence or confocal microscope. This detection process is also time-consuming because each element in the ASO array is imaged sequentially for a few seconds or more. In other words, the detection is not in real-time. In addition, the instrumentation required to read the DNA chips is very expensive, costing between US $100,000 and US $200,000 for a typical setup.
While some polymorphisms include changes of multiple base pairs, other polymorphisms only include the change of a single base pair, known as an “SNP.” For example, many of the mutations identified in the KVLQT1 gene are missense mutations involving a single, mismatched base pair. Traditionally, mismatched bases were distinguished by performing a hybridization reaction at a temperature below the melting temperature, Tm, of the homoduplex (hybrid of two wildtype oligonucleotides) but above the Tm of the heteroduplex (hybrid of wildtype and mutant oligonucleotides). However, the melting temperature of a given DNA duplex varies with its content of A•T base pairs. Therefore, it is difficult to find a temperature that is optimal for the hundreds or thousands of oligonucleotides immobilized to the DNA chip.
One solution to this problem is to add either tetramethylammonium (“TMA”) salts, such as TMA chloride, or betaine to the hybridization buffer. These compounds are thought to minimize the differences in melting temperature due to A•T content, thereby allowing all hybridization reactions to be performed at a single temperature that is optimal for distinguishing mismatched bases. However, high concentrations (1-2M) of these compounds are required, which makes them very viscous. This viscosity leads to manipulation problems and the high concentrations may interfere with enzymatic reactions. Another solution involves using modified nucleotides to either increase the stability of A•T base pairs or decrease that of G•C base pairs. Another variant of this approach is to add a few universal bases (5-nitroindoles) to the end of an A•T-rich oligonucleotide to increase its stability. Another solution is to allow the hybridization reaction to proceed to its maximum extent at a cold temperature (e.g., −20° C.) and then slowly ramp up the temperature of the DNA chip to 60° C. This allows an independent melting curve to be measured for every DNA duplex that has formed on the chip. While this approach is the most rigorous, it is also very slow and requires several hours to obtain a complete melting curve.
While hybridization techniques using ASO probes are used to screen for known mutations in the gene, an alternative technique is required to screen for mutations that have not been identified or cataloged. The current technique for detecting unknown mutations is fairly laborious and involves a technique called single-strand conformational polymorphism (“SSCP”). In this technique, PCR is used to amplify the region of interest, usually an exon. The PCR product is then denatured and run on an electrophoresis gel. In the single-stranded state, the nucleotide sequence of the PCR product affects its mobility, so an oligonucleotide containing a mutation migrates at a different rate on the gel than the wildtype sequence. The oligonucleotide containing the mutation is then isolated from the gel and sequenced to determine the position and composition of the mutation. However, the electrophoresis and sequencing steps are extremely time consuming.
An alternative for detecting unknown mutations in genes is sequencing by hybridization (“SBH”). In de novo SBH, a fragment of genomic DNA (usually 80-200 nucleotides in length) is exposed to a microarray of short oligonucleotides (usually 6 to 8 bases in length) that contain all possible sequence permutations. SBH has also been used to resequence a portion of the gene, of known sequence, that contains a genetic polymorphism in which a series of overlapping oligonucleotides is synthesized and immobilized on a microarray (or synthesized in situ on the chip). The sequence of each of these oligonucleotides is complementary to the gene of interest and is offset by one position relative to the preceding oligonucleotide in the series. Two strategies have been described for determining (or “calling”) the sequence of the base(s) that have been changed by the mutation. In the first calling strategy, each position in the gene of interest is probed by four different oligonucleotides that are 25 bases in length, each of which is substituted with one of the four nucleotides in the middle (13th) position.
The second calling strategy uses two types of oligonucleotide probes, a single series of overlapping capture oligonucleotides and a mixture of four different fluorescently-labeled sequencing oligonucleotides. Each of the fluorescently-labeled sequencing oligonucleotides contains a unique nucleotide at the 5′ position but is degenerate at the other four positions. Resequencing is accomplished by first hybridizing oligonucleotides derived from the gene of interest to the microarray and then adding the mixture of fluorescently-labeled sequencing oligonucleotides. The fluorescently-labeled sequencing oligonucleotides are typically too short to hybridize on their own, but may hybridize in a tandem fashion immediately adjacent to one of the capture oligonucleotides, forming a stable, but nicked, DNA duplex. Even though this nicked duplex has been shown to be thermodynamically stable, the capture and fluorescently-labeled sequencing oligonucleotides may also be ligated using polynucleotide ligase for improved stability.
Optical sensors, such as evanescent wave biosensors, are commonly used to detect various substances, or analytes, in diagnostic and research settings. For example, the BIACORE® biosensor, available from Biacore AB (Uppsala, Sweden), is based on surface plasmon resonance (“SPR”) and is used to monitor biomolecular interactions in real-time without the use of fluorescent or radio labels. Affinity Sensors (Cambridge, England), a division of Thermo BioAnalysis Corp., makes a similar system called IAsys® that uses a slightly different optical geometry referred to as “resonant mirrors.” Both of these systems respond to changes in an index of refraction in an evanescent wave. The changes occur when a ligand, with a refractive index greater than that of water, binds to an immobilized capture molecule on the surface of the sensor. Examples of such binding include soluble antigens binding to immobilized antibodies and single-stranded PCR products binding to immobilized oligonucleotides. The devices produced by Biacore AB and Affinity Sensors are mass sensors because the signals change in proportion to the mass bound within the evanescent field. Both Biacore AB and Affinity Sensors have modeled the kinetics of mass binding to the sensor and have determined the relationship between ligand concentration in bulk solution and binding rate.
Surface plasmon resonance and resonant mirror sensors represent a specialized application of a more general surface sensitive optical technique called attenuated total reflection (“ATR”) that preferentially interrogates sample bound to the solid/liquid interface via the evanescent wave. In most ATR geometries, the interrogating radiation is confined to a thick waveguide in which light propagates in a simple zig-zag pattern. To a first approximation, the interaction of the evanescent wave with a surface bound sample increases linearly with the number of reflections per centimeter (N) of the light at the waveguide-solution interface. This number may be calculated using the simple expression N=cot θ/2D, where D and θ are the waveguide thickness and mode propagation angle, respectively (Figure B.1). Thus, for a given surface optical measurement at a specified angle of reflection, a 1 μm thick glass waveguide may be 150 times more sensitive than a 150 μm thick glass coverslip, and 1000 times more sensitive than 1 mm thick glass microscope slide.
Evanescent wave biosensors also include fiber and planar waveguides, which are so thin that incoupled light no longer propagates as a simple ray of light. Instead, when the waveguide thickness is on the order of microns, the incoupled light forms constructive and destructive interference patterns. Guided modes are a discrete set of constructive interference patterns that allow light to propagate down the waveguide. In general, greater than 95% of the guided light is confined to the waveguide itself. The evanescent wave refers to the remaining 5%, or less, of light intensity that penetrates just a few tenths of a micron into the lower refractive index media adjacent to the waveguide surface.
Planar waveguides are known in the art and are of a generally planar shape comprising two planar surfaces spaced by a width. Different types of planar waveguide sensors are known in the art, including injection-molded thick-film waveguides and integrated optical thin-film waveguides (“IOW”). Planar waveguides are described in U.S. Pat. Nos. 5,512,495, 5,677,196, 5,846,842, and 6,222,619 (all issued to Herron et al.) and U.S. Pat. Nos. 5,832,165, 5,814,565 (all issued to Reichert et al.), the disclosures of which are hereby incorporated herein, in their entireties, by this reference.
Evanescent wave biosensors are designed to function with or without fluorescent labels. As mentioned previously, surface plasmon resonance and other label-free optical sensors respond to mass changes in the evanescent wave. However, mass sensors have at least two limitations over fluorescent sensors. First, a mass sensor responds to any molecule bound within the evanescent wave, whether it is bound specifically or non-specifically. For this reason, nonspecific binding (“NSB”) is a significant problem with mass sensors. Both Biacore AB and Affinity Sensors have devoted significant efforts to developing immobilization chemistries with low NSB. The second limitation is that mass sensors require a significantly larger sensing area to measure a given concentration of analyte than a fluorescent sensor because mass detection is less sensitive than fluorescent detection. Therefore, to detect low levels of an analyte, the sensitivity of fluorescence detection is preferred.
In contrast, detection in a fluorescent biosensor is accomplished by the specific binding of a fluorescently-labeled “tracer” molecule to the ligand-capture molecule complex. The specific binding is accomplished through an affinity interaction, including, but not limited to, the binding of soluble antigens to immobilized antibodies or single-stranded PCR products to immobilized oligonucleotide probes. Alternatively, a fluorescently-labeled analyte or ligand molecule binds directly to the immobilized capture molecule. This latter situation is preferable for nucleic acid hybridization assays because the fluorescent label is directly incorporated into the analyte molecule using PCR. In either of these cases, NSB is only an issue with the fluorescently-labeled molecule, rather than with any molecule that happens to be in the evanescent wave. Therefore, fluorescence is a preferable method of detection.
More specifically, optical biosensors are used to perform nucleic acid probe assays, also known as molecular diagnostics or MDx assays. It is known that the hybridization kinetics of heteroduplex DNA is slower and reach a lower steady-state value than that of homoduplex DNA. In addition, variability in the A•T content and oligonucleotide length affect the hybridization kinetics. It is possible to control for these factors by using pairs of wildtype and mutant probes and taking the ratio of the two hybridization rates (Rmut/Rwt), thereby normalizing for A•T content and oligonucleotide length.
While a majority of the biosensors known in the art are fluorescent fiber optic sensors or label-free surface plasmon resonance sensors, other label-free evanescent wave formats have been described including interferometry, diffractometry and evanescent-illuminated light scatter. Recently, the use of evanescent wave biosensors in clinical applications has been disclosed. In Nilsson et al. (Nilsson, P., B. Persson, A. Larsson, M. Uhlen and P. A. Nygren “Detection of mutations in PCR products from clinical samples by surface plasmon resonance” J Mol Recognit 10, 7-17 (1997)), surface plasmon resonance is used to detect the presence of the human tumor suppressor p53 gene in breast tumor biopsy material. SNPs in clinical DNA samples are detected by comparing the rate of hybridization of PCR products to the rate of hybridization of the wildtype. The PCR products, which contained mismatched bases, give reduced levels of hybridization relative to the wildtype.
In Pilevar et al. (Pilevar, S., C. C. Davis and F. Portugal “Tapered optical fiber sensor using near-infrared fluorophores to assay hybridization” Anal Chem 70, 2031-7 (1998)), 25 pM levels of Helicobater pylori RNA are detected using a fluorescent fiber optic sensor, showing that fluorescent evanescent wave sensors are capable of performing highly sensitive MDx assays.
Schneider et al., Clinical Chemistry, 43(9):1757-1763 (1997) discloses using a Hartman interferometer to detect real-time hybridization of nucleic acids. The Hartman interferometer is an optic sensor that uses a single planar wave of linearly polarized light to detect the hybridization of target nucleic acids to a complementary single-stranded probe. The assay is able to differentiate between sequences with a 4-base pair mismatch.
In Stimpson et al., Genetic Analysis: Biomolecular Engineering, 13:73-80 (1996), an optical waveguide is used to detect SNPs by monitoring the binding and dissociation kinetics of oligonucleotide complexes to oligonucleotide probes. The SNPs are detected by a signal produced by a selenium conjugate.
Jensen et al., Biochemistry, 36:5072-5077 (1997) discloses using hybridization kinetics to detect SNPs between nucleic acid mimics, such as peptide nucleic acid (“PNA”)-DNA and PNA-RNA duplexes. The SNPs are detected using surface plasmon resonance.
In Bianchi et al., Clinical and Diagnostic Virology, 8:199-208 (1997), surface plasmon resonance is used in a nucleic acid hybridization assay to detect mutations in HIV-1 genomic sequences. The assay uses hybridization kinetics and real-time monitoring to detect the mutations.
Abel et al., Anal. Chem. 68:2905-2912 (1996) discloses using nucleotide hybridization assays to detect small variations in nucleic acid sequences. The variations are detected by fluorescence using a fiber optic sensor.
Publication WO 99/47705 discloses using a planar waveguide in a nucleic acid hybridization assay to detect a target polynucleotide. The assay uses fluorescence to detect hybridization.
Publications WO 96/35940, WO 95/33197, and WO 95/33198 disclose assays that use at least one planar waveguide to quantitatively detect an analyte of interest in an opaque fluid. The assays use fluorescent dyes to determine nucleic acid hybridization and acquire data in real-time.
Thus, a need remains for an improved method of detecting SNPs using an evanescent wave sensor. A need further remains for a method of detecting SNPs that uses a planar waveguide in a fluorescence assay. The method may reduce assay time by monitoring fluorescence in real-time. A need also remains for a high-throughput genetic screening assay for detecting known and unknown mutations in a gene of interest.